Multi-omic cross-sectional cohort study of pre-malignant Barrett's esophagus reveals early structural variation and retrotransposon activity.

Barrett's esophagus is a pre-malignant lesion that can progress to esophageal adenocarcinoma. We perform a multi-omic analysis of pre-cancer samples from 146 patients with a range of outcomes, comprising 642 person years of follow-up. Whole genome sequencing reveals complex structural variants and LINE-1 retrotransposons, as well as known copy number changes, occurring even prior to dysplasia. The structural variant burden captures the most variance across the cohort and genomic profiles do not always match consensus clinical pathology dysplasia grades. Increasing structural variant burden is associated with: high levels of chromothripsis and breakage-fusion-bridge events; increased expression of genes related to cell cycle checkpoint, DNA repair and chromosomal instability; and epigenetic silencing of Wnt signalling and cell cycle genes. Timing analysis reveals molecular events triggering genomic instability with more clonal expansion in dysplastic samples. Overall genomic complexity occurs early in the Barrett's natural history and may inform the potential for cancer beyond the clinically discernible phenotype.

Longitudinal tracking of 97 esophageal adenocarcinomas using liquid biopsy sampling.

BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) is rapidly rising and has a 5-year survival rate of <20%. Beyond TNM (tumor-node-metastasis) staging, no reliable risk stratification tools exist and no large-scale studies have profiled circulating tumor DNA (ctDNA) at relapse in EAC. Here we analyze the prognostic potential of ctDNA dynamics in EAC, taking into account clonal hematopoiesis with indeterminate potential (CHIP). PATIENTS AND METHODS: A total of 245 samples from 97 patients treated with neoadjuvant chemotherapy and surgery were identified from the prospective national UK Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) consortium data set. A pan-cancer ctDNA panel comprising 77 genes was used. Plasma and peripheral blood cell samples were sequenced to a mean depth of 7082× (range 2196-28 524) and ctDNA results correlated with survival. RESULTS: Characteristics of the 97 patients identified were as follows: 83/97 (86%) male, median age 68 years (SD 9.5 years), 100% cT3/T4, 75% cN+. EAC-specific drivers had higher variant allele fractions than passenger mutations. Using stringent quality criteria 16/79 (20%) were ctDNA positive following resection; recurrence was observed in 12/16 (75%) of these. As much as 78/97 (80%) had CHIP analyses that enabled filtering for CHIP variants, which were found in 18/78 (23%) of cases. When CHIP was excluded, 10/63 (16%) patients were ctDNA positive and 9/10 of these (90%) recurred. With correction for CHIP, median cancer-specific survival for ctDNA-positive patients was 10.0 months versus 29.9 months for ctDNA-negative patients (hazard ratio 5.55, 95% confidence interval 2.42-12.71; P = 0.0003). Similar outcomes were observed for disease-free survival. CONCLUSIONS: We demonstrate in a large, national, prospectively collected data set that ctDNA in plasma following surgery for EAC is prognostic for relapse. Inclusion of peripheral blood cell samples can reduce or eliminate false positives from CHIP. In future, post-operative ctDNA could be used to risk stratify patients into high- and low-risk groups for intensification or de-escalation of adjuvant chemotherapy.

Characterization of stress processes of Phaffia rhodozyma stress-resistant mutant.

A carotenoid-less Phaffia rhodozyma mutant (MCP 325) exhibited significantly higher resistance to oxidative stressors such as menadione, H2O2 and K2Cr2O7 than its astaxanthin-producing parental strain (MCP 324). The absence of carotenoids in the mutant did not explain this phenomenon. The cause of the decreased superoxide, hydroxyl radical and glutathione contents, the increased peroxide concentration and the elevated specific activity of catalase under uninduced conditions may be a second mutation. Peroxide treatment induced specific catalase activity in the mutant but not in the parental strain. Regulation of these processes led to the result that, in spite of the mutations, the two strains exhibited the same multiplication rate and generation time.

Co-localization of galectin-1 with GM1 ganglioside in the course of its clathrin- and raft-dependent endocytosis.

Mammalian galectin-1 (Gal-1), a beta-galactoside-binding lectin has a prominent role in regulating cell adhesion, cell growth and immune responses. Downregulation of these biological functions may occur via internalization of Gal-1. In the present study we have investigated the mechanism and possible mediator(s) of Gal-1 endocytosis. We show that internalization occurs at a temperature higher than 22 degrees C in an energy dependent fashion. After one hour incubation Gal-1 localizes in the Golgi system within the cells, and then disappears without accumulation in degradation compartments, such as lysosomes. Based on their strong intracellular co-localization, two glycoconjugates, GM1 ganglioside and CD7 are implicated in the sorting of internalized Gal-1 into Golgi. Other known Gal-1 binding glycoproteins on T cells (CD2, CD3, CD43 and CD45) do not cointernalize with the lectin. Internalization of Gal-1 depends on its lectin activity and follows dual pathways involving clathrin-coated vesicles and raft-dependent endocytosis.

Effect of polar and non-polar carotenoids on Xanthophylomyces dendrorhous membranes by EPR.

The red yeast Xanthophyllomyces dendrorhous is one of the microbiological production systems for natural carotenoids. High-performance liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy (EPR) experiments were performed on X. dendrorhous membranes in order to study the effect of incorporation rates of different type of carotenoids. In the case of fluid-phase membranes, it was found that polar carotenoids, such as astaxanthin and cis-astaxanthin, increased the EPR order parameter and decreased the motional freedom and phase-transition temperature. In contrast the non-polar carotenoids beta-cryptoxanthin and beta-carotene decreased the EPR order parameter and increased motional freedom and phase-transition temperature. A noteworthy coherence was observed between the polarities of the strains and the phase-transition temperatures.

Fidelity of binding of the guanidinium nucleic acid (DNG) d(Tg)4-T-azido with short strand DNA oligomers (A5G3A5, GA4G3A4G, G2A3G3A3G2, G2A2G5A2G2). A kinetic and thermodynamic study.

Short strand DNA oligomers (A5G3A5, GA4G3A4G, G2A3G3A3G2, and G2A2G5A2G2) and the guanidinium (g) linked thymidyl nucleoside d(Tg)4-T-azido associate as triplexes. The melting temperatures, Tm, the association and dissociation kinetic and thermodynamic parameters and activation energies for the triplexes were determined by UV thermal analysis. The hypochromic shift and Tm for triplex formation increases with increase in concentration and decreases with the number of mismatches. The melting temperatures are between 35 and 55 degrees C in the range of ionic strength of 0.06-0.24 and decrease with increase in ionic strength at 100 deg/(ionic strength unit). The melting and cooling curves exhibit hysteresis behavior in the temperature range 5-95 degrees C at 0.2 deg/min thermal rate. From these curves, the rate constants and the energies of activation for association (k(on), E(on)) and dissociation (k(off), E(off)) processes were obtained. The second-order rate constants, k(on), for the triplex formation at 288 K are between 10 and 500 M(-2) s(-1). Values of k(on) increase with the decrease in the ionic strength. The first order rate constants for the dissociation, k(off), at 288 K are between 10(-6) and 40 x 10(-6) s(-1) and increase with increase in ionic strength. The energies of activation for the association and dissociation processes are in the range -22 to -9 kcal/mol and 8 to 29 kcal/mol, respectively. At 6.3 x 10(-5) M/base and at the physiological ionic strength (0.15-0.30) and below, the triplex structures formed with d(Tg)4-T-azido and A5G3A5 and GA4G3A4G have well-defined Tm values. The melting curves with G2A3G3A3G2 and G2A2G5A2G2 are very shallow with small hypochromic shifts denoting negligible binding at physiological ionic strength. Therefore, with the increase in the G content (mismatched base pairs) at a certain concentration (e.g., 6.3 x 10(-5) M/base), discrimination (change in fidelity) occurs in the formation and strength of binding of d(Tg)4-T-azido to d(pAn pGm) oligomers. The standard molar enthalpies for triplex formation have in general larger negative values at low ionic strength than at high ionic strength, indicating that at lower mu values the formation of triplexes of d(Tg)4-T-azido with d(pAn pGm) are more favorable. The values of deltaH(standard)(288) calculated from the activation parameters are between -17 and -49 kcal/mol, and the values of deltaG(standard)(288) are between -7.5 and -11.8 kcal/mol for A5G3A5, GA4G3A4G, G2A3G3A3G2, and G2A2G5A2G2, respectively. There is a linear relationship in the enthalpy-entropy compensation for the triplex melting thermodynamics.

A microgonotropen branched decaaza decabutylamine and its DNA and DNA/transcription factor interactions.

The central pyrrole of a site-selective DNA minor groove binding tripyrrole peptide 1 has been attached to a branched decaaza decabutylamine via a -(CH-2)3-NHCO-(CH2)-3 linker to provide the decaaza-microgonotropen (8). The decaaza decabutylamine moiety of 8 was designed to have a much greater affinity to the phosphodiester linkages of the backbone of DNA. Employing Hoechst 33258 (Ht) as a fluorescent titrant, the equilibrium constants for the binding for of 8 to the hexadecameric duplex d(GGCGCA3T3GGCGG)/d(CCGCCA3T3GCGCC) and to calf thymus DNA were determined. The log of the product of equilibrium constants (log Kl1Kl2) for 1:1 and 1:2 complexes formation at A3T3 is 17 (35 degrees C). Results of studies of the inhibition of the binding of several proteins to target DNA are discussed. Binding of the E2F1 transcription factor to its DNA target is 50% inhibited at approximately 2 nM concentration of 8.

A microgonotropen pentaaza pentabutylamine and its interactions with DNA.

The central pyrrole of a site-selective DNA minor groove binding tripyrrole peptide (1) has been attached to N-protected pentaazapentacosanoic acid (17) via a -(CH2)3-NHCO-(CH2)3- linker to provide 19, subsequent deprotection provided the pentaaza microgonotropen 4. The polyamine moiety of 4 reaches out of the minor groove and binds to the phosphate backbone of DNA. We find when employing Hoechst 33258 (Ht) as a fluorescent titrant to follow binding of 4 to the hexadecameric duplex d(GGCGCAAATTTGGCGG)/(CCGCCAAATTTGCGCC) and by 1H NMR titration of d(CGCAAATTTGCG)2 with 4 that the latter forms both 1:1 and 2:1 dsDNA complexes. Certain aspects of the structure of 4:d(CGCAAATTTGCG)2 complex derived via 1H NMR are discussed. The electrophoretic mobilities of phi X-174 DNA digested with HaeIII endonuclease restriction fragments complexed to 4 shows that the latter brings about a greater conformational change in the DNA fragments than observed previously with other microgonotropens.

NMR structure of d(CGCA3T3GCG)2:tren-microgonotropen-b:Zn(II) complex and solution studies of metal ion complexes of tren-microgonotropen-b interacting with DNA.

The solution structure of a 1:1 complex of zinc tren-microgonotropen-b [6b:Zn(II)] with d(CGCAAATTTGCG)2 has been determined by 2D nuclear Overhauser effect 1H NMR spectroscopy and restrained molecular modeling. The exchangeable and nonexchangeable proton resonances of d(CGCA3T3GCG)2:6b:Zn(II) indicate that the Zn(II) is interacting in the A+T-rich region of the dsDNA and the tren region of 6b, while 31P NMR shows interaction of the Zn(II) with the phosphate backbone. Proton chemical shift differences between d(CGCA3T3GCG)2:6b:Zn(II) and d(CGCA3T3GCG)2:6b are in agreement with the polyamino substituent of 6b [-(CH2)4N(CH2CH2)N-(CH2CH2NH2)2] forming a four-coordinated Zn(II) complex similar to that found in the X-ray structure of 'tren-chloride':Zn(II). The P9 and P10 phosphate oxygens that are held by hydrogen bonding to the tren substituent of 6b in the DNA:6b complex become ligands to the tren-complexed Zn(II) in DNA:6b:Zn(II). To do so there is a 2 A decrease in the adjacent phosphate-to-phosphate distance at the Zn(II) binding site. This motion brings about an increased bend of 14.6 degrees in the helical axis of d(CGCA3T3GCG)2:6b:Zn(II) compared to that found in d(CGCA3T3GCG)2:6b. Single stranded cleavage of linear DNA fragments was not observed in the presence of 6b and Fe(II), Co(II), Ni(II), Cu(II), Zn(II), La(III) or Ce(III); this is likely due to the metal ion being sequestered as in the structure of d(CGCA3T3GCG)2:6b:Zn(II) complex. Supercoiled DNA was susceptible to cleavage by 6b:Cu(II) in the presence of O2 and a reducing agent.

Stoichiometry and structure of complexes of DNA oligomers with microgonotropens and distamycin by 1H NMR spectroscopy and molecular modeling.

Dien-microgonotropen-c (5c), tren-microgonotropen-b (6b), and distamycin (Dm) bind the A.T-rich region of d(CGCAAATTTGCG)2 (oligo-12) and form 1:1 (5c and 6b) and 2:1 and 4:1 (Dm) complexes. At 1.75 mol ratio of Dm/oligo-12 the 4:1 complex starts to form and coexists with the 2:1 complex and the free double-stranded DNA. No 1:1 and 3:1 complexes were seen, implying a preferential dimeric binding mode of Dm to oligo-12. At 4:1 mol ratio of Dm/oligo-12 there is extensive exchange of the A.T imino protons with the solvent at the binding site. This is presumably due to the opening of the minor groove. Molecular modeling shows that four Dm molecules can fit in a tandem antiparallel way into the minor groove of oligo-12 by widening it to 16-17 A. On going from oligo-12 to the pseudosymmetrical hexadecamer oligo-16 [d(GGCGCAAATTTGGCGG).d(CCGCCAAATTTGCGCC)] the stoichiometry of binding of 5c changes from 1:1 to 2:1. Since oligo-12 and oligo-16 have the same A.T binding site this change in stoichiometry is due to the increase in the G.C terminal pairing. Hoechst 33258 displaces the two 5c molecules bound in the minor groove of oligo-16 at the A.T region. Marked exchange of A.T imino protons was seen in the case of (oligo-16).(Ht)2.